METHODS FOR DETECTION AND QUANTITATION OF SMALL RNAs

ABSTRACT

Improved methods that increase the specificity and sensitivity of detection of small RNAs, including miRNAs, using oligonucleotide primers and nucleic acid amplification, are provided. Reaction conditions that result in preferential decrease in cDNA synthesis of RNAs other than the small RNA molecules targeted for detection during miRNA tailing and reverse transcription reactions are described. Using these reaction conditions greater sensitivity and specificity of amplification of small RNAs including miRNAs is achieved.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/291,010, filed Nov. 4, 2008, now allowed, which application isincorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing 830109_(—)403C1_SEQUENCE_LISTING.txt. The text fileis about 2 KB, was created on Nov. 26, 2012, and is being submittedelectronically via EFS-Web herein.

BACKGROUND OF THE INVENTION

The methods disclosed herein relate to detecting and quantifying nucleicacids, especially small RNAs, including miRNAs and genes coding forsmall RNAs including but not limited to miRNAs. In the disclosurevarious reactions may be described in respect to miRNA, but it isunderstood by those skilled in the art that such reactions or reactionconditions would apply similarly to other small RNAs, of which miRNAsare an important subclass. More specifically, the present methods relateto novel reaction conditions under which a homopolymeric tail is addedto the 3′ end of a small RNA using poly-adenosine (poly(A)) polymerase(polyadenylation) and subsequently rendered into cDNA by reversetranscriptase under conditions that permit greater discrimination of atarget miRNA from non-miRNA contained in the same sample. These novelreaction conditions lead to greater specificity of addition ofnucleotides by both polymerases and greater specificity of detection ofthe small RNA being assayed. These reaction conditions allow forconvenient use of both poly(A) polymerase and reverse transcriptaseunder substantially the same ionic and buffer conditions, therebyavoiding undesired dilution of the sample or purification of products(with potential loss of desired materials) between the poly(A)polymerase reaction and the synthesis of a cDNA copy catalyzed byreverse transcriptase. The ability to forego all purification orsubstantial dilution of sample between these two reactions as well assubsequent reactions that measure miRNA amounts greatly increases theease of the assay, as well as its reproducibility, accuracy andsensitivity.

MicroRNAs (miRNA) are single-stranded RNA molecules of about 18 to 28,often about 22, nucleotides in length, which regulate gene expression.Other small RNAs, including piRNAs, snoRNAs, and small guide or sgRNAs,play essential biological roles, and their detection and quantitationare desired. Still other small RNAs may be discovered that playimportant biological roles as well. Herein a small RNA is defined as anRNA shorter than about 100 nucleotides in length. All miRNAs, piRNAs,sgRNAs, snoRNAs and snRNAs, as well as other RNAs having fewer than 100nucleotides are “small RNAs”. These RNAs are encoded by genes that aretranscribed from DNA, but the transcription products are not translatedinto protein (non-coding RNA). They are, in the case of miRNAs,processed from primary transcripts known as pri-miRNA to short stem-loopstructures called pre-miRNA and finally to functional miRNA. MaturemiRNA molecules generally are partially complementary to one or moremessenger RNA (mRNA) molecules, and their principal regulatory functionis to down-regulate gene expression by translation repression. They werefirst described in 1993 by Lee and colleagues and are today recognizedas important regulatory molecules in eukaryotic cells. MicroRNAs havebeen shown to play key roles in development, apoptosis, and cancer. Theyhave also been shown to be coded for by certain viruses.

Specific, sensitive and quantitative detection and assay of individualsmall RNAs and detection of miRNAs play an especially important role inbiomedical and biological research and promise to be of diagnostic andprognostic value in medical practice as well.

Notwithstanding their importance as regulatory molecules, small RNAsrepresent a small fraction of the RNA species in a eukaryotic cell.Because they are short, in the case of miRNAs, about 18-28 nucleotidesin length, or 18-50, or 18-100, and share homology or partial homology,with the genes they regulate, detection of specific miRNAs andespecially accurate quantitation of miRNAs present experimentalchallenges. Improvements to methods for the specific detection andquantitation of miRNAs will accelerate basic research. Improved assaysof miRNAs can prove of diagnostic or prognostic value in medicine.

One of the most widely used and effective ways to detect and quantifyRNAs is to produce a complementary DNA (cDNA) transcript of specificRNA, and then to amplify that DNA using the polymerase chain reaction(PCR) under conditions where the accumulation of the amplified productis monitored during the amplification, usually by means of fluorescentmolecules added to the PCR. This method is widely known as Real-TimeQuantitative RT-PCR(RT-qPCR). Improved methods to assay specific miRNAsby RT-qPCR are desired.

When assaying for species as low in abundance as miRNA, the specificityof detection and accurate quantitation are usually higher prioritiesthan absolute analytical sensitivity. Accordingly, modifications tostandard processes that increase specificity and accuracy ofquantitation, even if they result in minimal or modest losses ofabsolute analytical sensitivity, are likely to be preferred.

In light of the vast abundance of RNAs other than the specific small RNAbeing assayed, or especially in the case of assaying for a specificmiRNA, non-specific amplification of other sequences can compromise thesensitivity, specificity, and precision of an RT-qPCR assay for a smallRNA, especially an miRNA.

One approach to the specific amplification of miRNAs, but alsoapplicable to other small RNAs, is that used by Ro et. al. (BiochemBiophys Res Commun (2006) 351 (3) 756-763) which involves addition tothe 3′ prime end of the miRNA to be amplified (the target miRNA) apoly(A) tail of greater than about 20 base pairs. This can be done usingpoly(A) polymerase and ATP; this lengthens the miRNA molecule in orderto facilitate subsequent cDNA synthesis and detection. Anoligonucleotide, with the 5′ sequence region matching a universal qPCRprimer sequence and the 3′ sequence region being approximately 20 d(T)bases and complementary to the poly(A) tail, is hybridized to the 3′ endof the target miRNA to provide a template which can be used by a reversetranscriptase to produce a cDNA molecule capable of being amplified byPCR. This cDNA contains a region complementary to a specific miRNA, acentral region of approximately 20 T's (on one strand and the samenumber of A's on the other) and a third region that contains anarbitrary sequence that can serve well as a site for specific priming bya PCR primer, i.e., a universal primer. Note, that cDNAs made fromdifferent miRNAs, will have all have a common universal primer sequence.However, this reaction is not absolutely specific and other RNAs presentin the sample and such other RNAs having been present in vast abundancerelative to the targeted miRNA in the cells, from which the miRNA isprepared, in spite of earlier purification steps taken to eliminatethem, may have poly(A) tails added to them. Some of these non-miRNAs mayalso serve as template for reverse transcriptase and receive a universalprimer sequence. Reduction in non-specific tailing of RNAs andgeneration of RNAs other than miRNAs that contain universal primersequence is desired. All of the advantages described above pertaining tomiRNAs also apply to other small RNAs.

Purified poly-adenosine polymerase has been commercially available foryears to artificially produce poly(A) tails on RNA molecules in vitro.Recently this enzymatic activity has been utilized as the first step inan miRNA cDNA generation system to increase the length of the miRNA forincreased PCR performance and to provide a uniform primer binding siteto initiate cDNA synthesis.

Purified reverse transcriptases have been used to produce DNA copies ofRNAs in vitro following well accepted reaction conditions and protocols.This reaction has become a standard process used in thousands ofresearch laboratories. Several manufacturers of research products havemarketed kits containing premixed buffers for customer use. Like mostenzymes that catalyze the polymerization of nucleotide triphosphatesinto DNA or RNA using a template nucleic acid as a template, reversetranscriptases have standard reaction conditions in which theconcentration of magnesium ions is maintained between about 2 millimolarand 5 millimolar. Biochemists experienced in the optimization of reversetranscriptase reaction in vitro are especially careful to optimize theconcentration of magnesium ions within this overall range, about 2millimolar to about 5 millimolar. The Applicants are unaware of anypublished reports of reaction conditions for reverse transcription invitro carried out with a magnesium concentration above 10 mM.

The concentration of cations, particularly divalent cations such asmagnesium and manganese are known to affect the secondary structure ofnucleic acids, particularly single stranded nucleic acids such as mostRNAs found in cells, including human cells (Bukhman and Draper, J. Mol.Biol. (1997) 273, 1020-1031) Nearly all RNAs that are abundant in cells,especially, but not only ribosomal RNAs, have considerable secondarystructure that can be affected by the ionic environment, especially theconcentration of cations such as magnesium and manganese. While methodsare known that result in the preferential purification of small RNAs,miRNA purified using these methods can be heavily contaminated withlarger RNAs, with other small RNAs, and degradation fragments of largeRNAs such as ribosomal RNAs. In any sample of RNA, even one enriched forshorter RNAs, any particular miRNA will be a minor constituent in thesample. Thus, there is a need in the art for improved methods whichallow selective amplification of one or more target small RNA moleculesin a sample.

SUMMARY OF THE INVENTION

The invention comprises a new and improved method for synthesis of acDNA that contains the sequence of an miRNA or other small RNA that canbe amplified using standard nucleic acid amplification methods such asthe Polymerase Chain Reaction is disclosed. The method disclosed hereinprovides higher specificity of cDNA synthesis from small RNAs, whilesimultaneously permitting experimenters to carry out the two keyenzymatic reactions necessary for this synthesis under substantially thesame reaction conditions, conditions that include the presence ofdivalent cations at concentrations from 10 millimolar and 80 millimolar.When these reactions conditions are used as part of an assay for a smallRNA, especially for an miRNA, greater specificity and sensitivityresults.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an exemplary first derivative of the thermal dissociationcurve for a miRNA specific qPCR reaction in which there is goodspecificity of the resulting products, as indicated by a single majorpeak. FIG. 1B is an exemplary “Bad Assay”, reflected in a thermaldissociation curve whose shape results from an unacceptable level ofnon-specific amplification as indicated by more than one major peak.

FIGS. 2A-2D show that, when a high input of hsa-mir-658 RNA (120,400copies) is added (FIGS. 2A and 2B) against a background of small RNAfrom 293 cells, low Mg++ concentration (3 mM) in the tailing and reversetranscription reactions results in substantial amplification ofnon-specific targets and at this hsa-mir-658 RNA input higher Mg++concentrations (20 mM, 50 mM and 70 mM) yield only amplification of thedesired target (peak at Tm 79° C.). At 100 fold lower input hsa-mir-658RNA (1204 copies) (FIGS. 2C and 2D) only concentrations of 50 mM and 70mM Mg++ in the tailing and reverse transcription reactions resulted inamplification of only the desired target, though use of 20 mM Mg++resulted in less amplification of non-specific RNA than use of 3 mMMg++.

FIG. 3 shows the real-time PCR (qPCR) results (Ct values) for a miR-658specific assay from 10-fold serial dilutions of the hsa-mir-658synthetic RNA oligonucleotide into 100 ng small RNAs from 293H cells inwhich four different concentrations of Mg++ ions (3, 20, 50 and 70 mM)were used during the poly(A) tailing and reverse transcriptasereactions. In order to simulate a uniform complex nucleic acidbackground in the miRNA RT-qPCR process, a constant amount (10 ng/μl) of293H cell small RNA was included in every sample and thereforecontributes a uniform background as seen in the low copy number datapoints. This background signal decreases with increasing Mg++ levels sothat it is 1663-fold lower when 70 mM Mg++ is used in the enzymaticreactions than when 3 mM Mg++ is used. The linearity of the signal withdecreasing target input continues to lower target inputs when the higherMg++ concentrations are used thereby providing a more sensitive assayand one which can be quantitative at lower target concentrations.

FIGS. 4A-4B show the benefit of performing the cDNA synthesis of smallRNA targets under conditions of high Mg (FIG. 4B), which extends to theuse of human total RNA samples as a target source material as comparedunder conditions of low Mg (FIG. 4A). This is indicated by the overallincrease in Ct values obtained from qPCRs across 376 assays in a humanmiRNA assay library in the dissociation curves. Ct values greater than35 are considered to be below the level of quantitative detection bythis qPCR.

FIG. 5 shows the thermal dissociation curves for four additional miRNART-qPCR assays for hsa-miR-10a (FIGS. 5A and 5B), hsa-miR-346 (FIGS. 5Cand 5D), hsa-miR-504 (FIGS. 5E and 5F), and hsa-miR-555 (FIGS. 5G and5H), where “Bad Assay” results are obtained under 3 mM Mg conditions(FIGS. 5A, 5C, 5E and 5G) and good results are obtained using 70 mM Mg(FIGS. 5B, 5D, 5F and 5H).

DETAILED DESCRIPTION OF THE INVENTION

In the present context, miRNA is referred to because it is an archetypeof the small RNAs for which the present methods are especially wellsuited; however, all the advantages pertaining to the present methodsfor detection of miRNA are similarly applicable to detection of allshort RNAs. Notwithstanding the well accepted range of magnesium ionconcentration appropriate for poly(A) tailing and reverse transcription,alternative reaction conditions, in which magnesium ions are included atsignificantly higher concentration than 5 millimolar are disclosedherein, and such conditions have been found to be compatible withefficient catalysis by reverse transcriptase. Surprisingly, theseconditions increase the specificity of addition of poly(A) tails to adesired target miRNAs while minimizing addition of poly(A) tails tosmall RNA molecules longer than miRNAs.

MicroRNAs (miRNA) are single-stranded RNA molecules of about 15 to about50 nucleotides, or up to 75 or up to 100 nucleotides, especially about18 to 28 nucleotides in length, which regulate gene expression. Othersmall RNAs, include piRNAs and snoRNAs. piRNAs are Piwi-interactingRNAs; they are expressed in mammalian testes and somatic cells and theyform RNA-protein complexes with Piwi proteins. The piRNAs function ingene silencing of retrotransposons and other genetic elements in germline cells. Typically, piRNAs are about 26-31 nucleotides long.Generally the snoRNAs are in the range of about 70 to about 185nucleotides in length. However, the specificity for amplification isincreased with small RNA molecules up to about 100 nucleotides inlength.

Insofar as miRNAs and other small RNAs are short in length, on the orderof a typical primer used for extension of a template, little flexibilityis available in terms of sequence selection for a PCR primer. Theshorter the RNA, the less flexibility of primer selection is generallyavailable. This contrasts with larger RNAs such as mRNAs where multipleprimers can be considered and tested, and those that exhibitunacceptably low priming efficiency or specificity may be avoided. Inthe case of an miRNA and other small RNA molecules, few primer optionsare available, and all (or most) of them share considerable sequencehomology or overlap with one another. If a priming sequence exhibitsunacceptable priming specificity (FIG. 1), the experimenter must findapproaches other than alternative priming sites to increase assayspecificity.

In most applications in which one desires to add a poly(A) region to the3′ end of a collection of RNAs, optimal performance entails having theenzyme add poly(A) to all of the RNAs in the collection and to do sowith substantially the same efficiency for each RNA in the sample. Inthe situation in which one wished to detect miRNAs or other small RNAsthat are represented in the mixture at low levels, conditions thatpreferentially add poly(A) to miRNAs or other small RNAs are desirable,even if these conditions lower the efficiency of tailing for the desiredmiRNA or small RNA.

It was found that if the concentration of magnesium ions present in boththe poly(A) tailing and reverse transcription reaction mixture is about50 to 70 mM, a concentration far higher than has been previously used invitro for both enzymes, the desired tailing reaction proceedsefficiently and the specificity of addition of poly(A) tails to thedesired miRNAs is enhanced (FIG. 2). At the same time, non-target RNAs,which are also potential substrates for the enzymes, take oncharacteristics under high magnesium conditions that reduce theefficiency of tailing their 3′ end. Because the specificity of additionof poly(A) tails is of high importance, the specificity and sensitivityof detection and the precision of quantitation of specific miRNAs orother small RNA is increased. The optimal concentration for enhancingthe specificity of poly(A) tailing with poly(A) polymerase may varyaccording to the miRNA or other small RNA an investigator desires todetect and quantify, but absolute optimization may not be required. Thepresent inventors have concluded that a concentration of magnesium ionsin the range of 50 to 70 mM is suitable for all miRNAs or other smallRNAs, providing significantly increased specificity and sensitivity ascompared to reaction conditions in which a magnesium concentration is inthe range of 2 mM to 5 mM, the range heretofore typically used for invitro reverse transcription. Furthermore, it has been observed that thegeneration of cDNA through the use of reverse transcriptase can also becarried out at a magnesium ion concentration in the range of about 50 mMto 70 mM, a concentration that has never been reported as suitable forpolymerization by reverse transcriptase. Insofar as reversetranscription to yield cDNA immediately follows tailing by poly(A)polymerase and it is not advantageous to dilute the reaction, theability to carry out reverse transcription at a magnesium ionconcentration in the range of 50 to 70 mM provides significant benefitin terms of product yield, experimental efficiency and convenience (FIG.3).

While divalent manganese cations have the beneficial effect on thereverse transcriptase step, the use of manganese is not preferredbecause of the inhibitory effects on certain polymerases (especially Taqpolymerase) used in the amplification reaction. Thus, purification awayfrom the manganese is required if the Taq (or equivalent) polymerase isto be used.

Without wishing to be bound by theory, it is believed that the mechanismfor the observed effect of magnesium at a concentration from about 50 mMto about 70 mM, is the stabilization of secondary structure and internalbase pairing in RNAs which are longer than miRNAs, which are present inthe preparation despite efforts to eliminate them by prior purification.This increased stability of secondary structure and intramolecular basepairing is believed to reduce the availability of the 3′ RNA terminal tooccupy the catalytic site of the poly(A) polymerase and to reduce theability of the reverse transcriptase primer oligonucleotide to hybridizeto RNAs other than the miRNA or other small RNA being targeted, butwhich share partial homology to the targeted miRNA or other small RNA.Being short, in the range of 18 to 28 bases in length, miRNAs havelittle ability to form secondary structure even with the stabilizingeffects of elevated magnesium concentration. Other small RNAs areexpected to have similarly reduced ability to form secondary structure.No significant reduction in tailing of miRNAs has been observed usingpoly(A) polymerase at magnesium concentrations in the range of 60 mM to70 mM as compared to tailing at magnesium concentrations in the commonlyused range of 2 mM to 5 mM.

While most users of reverse transcriptase use a reverse transcriptaseencoded by Mouse Moloney Leukemia Virus or a genetically engineeredmutated form of this enzyme, other reverse transcriptases may be usedand the improved reaction conditions comprising elevated magnesiumconcentration confer the same or similar advantages if these otherreverse transcriptases are used.

By inhibiting hybridization by the DNA oligonucleotide primer, theelevated magnesium reduces reverse transcription of these longer,non-targeted RNAs.

Increasing the concentration of magnesium ions to preferentiallystabilize the secondary structure of non-mi-RNAs has been used becausemagnesium is known to be compatible with efficient catalysis by reversetranscriptases. Other cations, especially other divalent cations, suchas manganese, may provide similar improved specificity of tailingwithout unacceptably reducing the catalytic efficiency of reversetranscriptase.

A key measure of the efficiency and specificity of the tailing reactionis the frequency at which non-specific amplification becomes so high asto render results unreliable. In Table 1 multiple reactions (468different human miRNA qPCR assays) were carried out using a range ofmagnesium chloride concentrations in the tailing reaction and qPCR. Twodifferent input RNA levels were also used. Reactions conducted using 50mM or 70 mM MgCl₂ consistently showed a lower percentage of reactionsexhibiting unacceptable high levels of non-specific amplification. “BadAssays” as a descriptor in Table 1 are ones in which the thermaldissociation curve of the amplification products indicated non-specificamplification as evidenced by more than one peak. Four different celllines were used as a source of miRNA for all 468 assay and a “Bad”result from even a single cell line was tallied as a “Bad” result in thetable. An example of the melting curve of an assay that is not a “BadAssay” is shown in FIG. 1A and an example of a “Bad Assay” is shown inFIG. 1B.

In addition, the results in Table 1 demonstrate that the MgCl₂ effectoccurs only during the cDNA synthesis step and not the qPCR steps. Thisis evident from the fact that the 3 mM “plus” column results, where thetailing and RT were performed at 3 mM but the qPCR performed at thelevel equivalent to 70 mM, match the standard 3 mM results and not the70 mM results.

TABLE 1 Tailing/RT qPCR results for a human miRNA assay library testedon 4 human cell lines under different conditions of MgCl2 and RNAtemplate input. 3 mM 3 mM 50 mM 70 mM 3 mM “plus” 70 mM [MgCl₂] (50 ng(50 ng (50 ng (100 ng (100 ng (100 ng (RNA input) RNA) RNA) RNA) RNA)RNA) RNA) “Bad” Results 271 213 170 241 244 152 out of 468 Assays

All references cited herein are hereby incorporated by reference to theextent they are not inconsistent with the present disclosure, and forexample, a reference that is partially inconsistent is incorporated byreference except for the partially inconsistent portion of thereference. Those references reflect the state of the art and the levelof skill in the relevant art. References cited herein are incorporatedby reference herein in their entirety to indicate the state of the art,in some cases as of their filing date, and it is intended that thisinformation can be employed herein, if needed, to exclude (for example,to disclaim) specific embodiments that are in the prior art. Forexample, when a compound is claimed, it should be understood thatcompounds known in the prior art, including certain compounds disclosedin the references disclosed herein (particularly in referenced patentdocuments), are not intended to be included in the claim.

Although the description herein contains certain specific information,these should not be construed as limiting the scope of the invention butas merely providing illustrations of some of the presently preferredembodiments of the invention. For example, thus the scope of theinvention should be determined by the appended claims and theirequivalents, rather than by the examples given.

When a compound or concentration in a method is claimed, it should beunderstood that compounds and concentrations known in the art includingthose disclosed in the references disclosed herein are not intended tobe included. When a Markush group or other grouping is used herein, allindividual members of the group and all combinations and subcombinationspossible of the group or range are intended to be individually includedin the disclosure.

Every formulation or combination of components described or exemplifiedcan be used to practice the invention, unless otherwise stated. Specificnames of compounds and enzymes are intended to be exemplary, as it isknown that one of ordinary skill in the art can name the same compoundsand enzymes differently. When a compound or enzyme is described hereinsuch that a particular isomer, enantiomer or isoform of the compound orenzyme is not specified, for example, in a formula or in a chemicalname, that description is intended to include each isomer, enantiomer orisoform of the compound or enzyme described individual or in anycombination.

One of ordinary skill in the art will appreciate that methods, enzymesor starting materials, other than those specifically exemplified can beemployed in the practice of the invention without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, starting materials, synthetic methods, andenzymes are intended to be included in this invention. Whenever a rangeis given in the specification, for example, a concentration range, atemperature range, a time range, or a composition range, allintermediate ranges and subranges, as well as all individual valuesincluded in the ranges recited are intended to be included in thedisclosure.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, is understoodto encompass those compositions and methods consisting essentially ofand consisting of the recited components or elements. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, limitation or limitations which is notspecifically disclosed herein.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art.

The invention may be further understood by the following non-limitingexamples.

EXAMPLES Example 1

Cultured mammalian cells (MCF7, 293H, SKBR3 or Jurkat) were harvestedinto a pellet containing up to 5×10⁶ cells per tube. One ml of Trizol®(Invitrogen, Carlsbad, Calif.) was added, mixed and allowed to incubateat room temperature (RT) for 5 minutes. 200 μl of chloroform was added,mixed and centrifuged for 5 minutes at 12,000×g at 4° C. 400 μl of theupper aqueous phase was transferred to a new tube and mixed with 215 μlethanol. This mixture was applied to a NucleoSpin® RNA II spin column(Macherey-Nagel, Duren, Germany) and centrifuged for 30 seconds at11,000×g at RT. The eluate from the column, which contains small RNA,was mixed with 750 μl ethanol and applied to a second spin column asdescribed above. Under the condition of greater ethanol concentration,the small RNA was retained on the column. The column was washed byadding 200 μl wash buffer (1 part RA3 Buffer (Macherey-Nagel, Duren,Germany) plus 4 parts 100% ethanol) and centrifuging for 30 seconds at11,000×g at RT. This was followed by a second wash with 250 μl 70%ethanol and centrifugation for 3 minutes at 11,000×g at RT. The smallRNA enriched sample was eluted from the spin column with 40 μl water andcentrifugation for 1 minute at 11,000×g at RT.

RNA tailing and reverse transcription into cDNA was performed in onesimultaneous reaction using 50 ng to 400 ng of small RNA sample. Thetypical 10 μl reaction mixture consisted of 0.8 μM reverse transcriptionDNA primer (DNA Sequence #1), 10 mM tris-HCl, pH 8.0, 75 mM KCl, 10 mMDTT, 500 μM ATP, 2.5 mM each dGTP, dATP, dTTP & dCTP, 1 U E. coliPoly(A) Polymerase (New England Biolabs), 160 U M-MLV reversetranscriptase (Promega) and 70 mM MgCl₂. However, depending on theexperiment other MgCl₂ concentrations may have been used, specifically 3mM, 20 mM and 50 mM. After mixing the components and brieflycentrifuging, the reaction was incubated at 37° C. for 30 minutesfollowed by 95° C. for 5 minutes. The resulting sample cDNA was thenplaced on ice and diluted with 90 μl nuclease-free water (10 fold) sothat 1 μl (1/100) was used as the template for each individual 25 μl PCRreaction.

The amount of specific miRNA sequences present in the samples wasquantified by SYBR® Green real-time PCR. Individual samples were assayedby combining 12.5 μl 2×SYBR® Green PCR Master Mix (ABI) with 1 μl cDNAsample, 1 μl PCR primer mixture containing 10 μM each, specific forward(DNA Sequences #2 through #6) and universal reverse primer (DNA Sequence#7) and 9.5 μl nuclease-free water. Real-time PCR thermal cycling anddetection was performed on either an ABI 7500, ABI7900 or StratageneMx3005P instruments. Thermal cycling conditions were programmed to be 10minutes at 95° C. then 40 cycles of 15 seconds at 95° C., 30 seconds at60° C. and 60 seconds at 72° C. and concluding with the instrumentspecific thermal dissociation sub-program.

Using the instrument's software and a consistent selection ofmeasurement variables, Ct values were determined. Once control reactionresults were assessed for a consistent quality criteria, relative miRNAexpression measurements could be obtained by the ΔΔCt calculation method(Livak 2001) using the average Ct of four constitutively expressed smallRNA assays to normalize the data within each sample.

In addition, assays were evaluated for specificity of the PCR byanalyzing the first derivative of the thermal dissociation profiles fromthe final reaction products. This method of analysis allowed thedetermination of the extent to which RNAs other than the intended targetwere tailed and then amplified in the PCR (FIG. 1). The various productsexhibit melting peaks along the temperature axis, and the apex of eachpeak and its symmetry are indicative of the purity of the resultingproduct. The melting profiles of the products generated from thereaction in which tailing and reverse transcription was carried out atmagnesium ion concentrations of 50 mM and 70 mM reflected the greaterspecificity of tailing than those carried out at lower magnesiumconcentrations (FIG. 2) and indicated that less non-specific tailing andsubsequent PCR amplification resulted when these conditions of elevatedmagnesium were used.

hsa-miR-658 Amplification

For the experiments shown in FIG. 2A, 120,400 copies of synthetichas-miR-658 were spiked into a sample containing 100 ng 293H small RNA.Polyadenylation and reverse transcriptase reactions were run intriplicate in the presence of 3 and 70 mM MgCl₂. FIG. 2B shows theresults for reactions run in the presence of 20 and 50 mM MgCl₂. Thereaction products were analyzed by PCR. An oligonucleotide of thesequence set forth in SEQ ID NO:1 was used as the reverse transcriptionprimer for poly(A) tailed RNA, the miRNA specific primer had thesequence set forth in SEQ ID NO:6 and was used with the reverseuniversal primer of SEQ ID NO:7.

For amplifications of hsa-miR-10a, shown in FIGS. 5A and 5B, theexperiments were carried out as described above with 3 or 70 mM Mgcation, but using the oligonucleotide of SEQ ID NO:3 as forward primer.

For amplifications of hsa-miR-346, shown in FIGS. 5C and 5D, theexperiments were carried out as described above with 3 or 70 mM Mgcation, but using the oligonucleotide of SEQ ID NO:2 as forward primer.

For amplifications of hsa-miR-504, shown in FIGS. 5E and 5F, theexperiments were carried out as described above with 3 or 70 mM Mgcation, but using the oligonucleotide of SEQ ID NO:4 as forward primer.

For amplifications of hsa-miR-555, shown in FIGS. 5G and 5H, theexperiments were carried out as described above with 3 or 70 mM Mgcation, but using the oligonucleotide of SEQ ID NO:5 as forward primer.

All sequences are written in 5′ to 3′ orientation. All miRNA accessionnumbers and sequences used for primer design are from the publicdatabase, miRBase, v10.0 (internet address,microrna.sanger.ac.uk/sequences/).

1) Reverse transcription (RT) primer for poly(A) tailed RNA:SEQ ID NO: 1 GTGCAGGGTCCGAGGTTCACTATAGGTTTTTTTTTTTTTTTTTTTTTTTTVN,where V is G, A or C and N is G, A, T or C2) Forward PCR primer for hsa-miR-346 (MIMAT0000773): SEQ ID NO: 2TGTCTGCCCGCATGCCTGCCTCT,3) Forward PCR primer for hsa-miR-10a (MIMAT0000253): SEQ ID NO: 3TACCCTGTAGATCTGAATTTGTG,4) Forward PCR primer for hsa-miR-504 (MIMAT0002875): SEQ ID NO: 4ACCCTGGTCTGCACTCTATCAA,5) Forward PCR primer for hsa-miR-555 (MIMAT0003219): SEQ ID NO: 5GGTAAGCTGAACCTCTGATAA,6) Forward PCR primer for hsa-miR-658 (MIMAT0003336): SEQ ID NO: 6GGCGGAGGGAAGTAGGTCCGTTGGT,7) Universal reverse PCR primer for cDNAs generated from theRT primer above: SEQ ID NO: 7 GTGCAGGGTCCGAGGT,

1.-14. (canceled)
 15. A method for preparing a cDNA copy of a small RNAmolecule, comprising: (a) providing a small RNA from a biologicalsample, wherein said RNA is from 18 to 28 nucleotides in length; (b)incubating the small RNA with an enzyme capable of catalyzing theaddition of nucleotides at the 3′ end of the small RNA in the presenceof a single ribonucleotide triphosphate selected from the groupconsisting of ATP, GTP, UTP, and CTP and at a final concentration ofdivalent magnesium cation between 20 millimolar and 80 millimolar in areaction to add nucleotides to the small RNA to generate a tailed smallRNA; (c) annealing a DNA primer to the tailed small RNA whereby the DNAtemplate extends from the 3′ end of the tailed small RNA, therebyproviding a single stranded region of DNA that may be used to directpolymerization of deoxyribonucleotide triphosphates; and (d) incubatingthe annealed tailed small RNA and DNA primer in the presence of reversetranscriptase and deoxyribonucleotide triphosphates and at a finalconcentration of divalent magnesium cation between 20 millimolar and 80millimolar under conditions allowing reverse transcription into cDNA andamplification of the annealed tailed small RNA to produce anamplification product.
 16. The method of claim 15, wherein the enzymeused in step (b) is Escherichia coli Poly(A) polymerase.
 17. The methodof claim 15, wherein polymerization in step (c) is catalyzed by is MMLVreverse transcriptase.
 18. The method of claim 15, wherein the steps(b), (c) and (d) are performed concurrently in a single reactionmixture.
 19. The method of claim 15, further comprising the step ofquantifying the amplification product of step (d).
 20. The method ofclaim 15, further comprising the step of detecting the amplificationproduct of step (d).
 21. The method of claim 18, further comprising thestep of quantifying the amplification product of step (d).
 22. Themethod of claim 18, further comprising the step of detecting theamplification product of step (d).
 23. The method of claim 15, whereinthe final concentration of divalent magnesium cation in steps (b) and(d) is between 30 millimolar and 80 millimolar.
 24. The method of claim15, wherein the final concentration of divalent magnesium cation insteps (b) and (d) is between 40 millimolar and 80 millimolar.
 25. Themethod of claim 15, wherein the final concentration of divalentmagnesium cation in steps (b) and (d) is between 50 millimolar and 80millimolar.
 26. The method of claim 15, wherein the final concentrationof divalent magnesium cation in steps (b) and (d) is between 70millimolar and 80 millimolar.